Growth Kinetics of CLL in the Blood

CLL is a clonal expansion of leukemic cells that results from the slow and progressive accumulation of ineffectual long-lived lymphocytes arrested in G0. The notion that CLL is an accumulative disorder dates back many decades ago (48,49). In the absence of significant cell death (see below), the slow pace of the disease indicates a relatively low proliferative capacity. This slow proliferation has been repeatedly documented using a variety of methods to determine cell cycle kinetics, both in vivo and in vitro. In early studies, the classical approach to the analysis of cell proliferation was based on the determination of thymidine incorporation into dividing cells. Theml et al. (50), using continuous infusion of 3H-thymidine and autoradiography in two patients with CLL, demonstrated that cell production was low and that the vast majority of the lymphocytes in the blood were long lived, with a turnover time longer than 1 year. Although lymphocyte proliferation was virtually absent in the blood and very low in the marrow, it was relatively high in the enlarged nodes. The exchange of lymphocytes seems partially intact between lymph nodes and blood but impaired between marrow and blood. Also, there is a reduced capability of CLL lymphocytes to recirculate from blood to blood via the lymph node and the thoracic duct (51). The disturbance of exchange of cells between the intra- and extravascular pools in CLL could explain some of their clinical manifestations (52,53). In vitro 3H-thymidine LI of marrow leukemic cells determined at diagnosis in CLL also demonstrated little labeling of leukemic lymphocytes, with a median LI as low as 0.05% (54). This initial LI was unrelated to age, sex, absolute number of circulating lymphocytes, degree of marrow lymphocytosis, or clinical staging.

The low proliferative capacity of CLL has also been demonstrated by other techniques. Using acridine orange as a fluorescent dye, Andreeff et al. (55) showed that CLL cells have diploid DNA, very low proliferation, and a low RNA content, similar to that found in normal B-cells. Likewise, de Melo et al. (56) demonstrated a very low percentage of proliferating cells by Ki-67 staining (1.4%).

Applying double-color immunofluorescence methods to determine BrdU incorporation and the expression of the nuclear proliferation-associated antigen, Ki-67, together with the pheno-typic profile of the cells, Stephenson et al. (57) also showed that unstimulated B-CLL cells are primarily in G0, like to peripheral blood lymphocytes. Lin et al. (58) measured PCNA and BrdU incorporation as surrogate markers for proliferation and found a very low level of proliferation in CLL. Even PLL, a process characterized by the presence of numerous circulating large cells with immunophenotypic features reminiscent of CLL, demonstrated only slightly higher prolif-erative potential, either by S-phase (59) or Ki-67 analysis (56).

The reasons for the low proliferative capacity of CLL cells, even after stimulation by a variety of polyclonal B-cell activators, are not completely understood, although it is likely that alteration in the expression of genes that participate in cell cycle control and progression may be in part responsible (60). Of interest was the observation that cases with trisomy 12 were associated with a higher percentage of Ki-67-positive cells and that most Ki-67-positive cells were trisomic for chromosome 12, as determined by fluorescence in situ hybridization (FISH) analysis (61). Also, thymidine uptake following mitogenic stimulation was significantly greater in CLL cells with an extra chromosome 12 than in those with normal karyotypes (62). These findings suggest that growth control mechanisms in CLL may reside at this chromosome location, at least in some cases.

2.2.2. Clinical Correlation

Although the circulating cells in most patients with CLL demonstrate little capacity to grow, some evidence of more active proliferation may be observed in some cases. This variability in growth capacity has been correlated with clinical variables to determine whether it has potential prognostic value. In one study, the initial in vitro 3H-thymidine labeling in marrow leukemic cells did not show prognostic significance (54). Likewise, in another study, relative thymidine uptake (radioactivity per 103 lymphocytes) did not prove to be a useful prognostic parameter (63). However, the latter authors showed that an absolute higher in vitro uptake of thymidine (by leukocytes in a standard volume of peripheral blood) was associated with a higher lymphocyte count, a more advanced stage, greater frequency of functional impairment, and shorter survival, suggesting that thymidine uptake by circulating leukocytes in CLL provides useful prognostic information. Similarly, Simonsson et al. (64) demonstrated a strong correlation between proliferation index (3H-thymidine uptake-based LI x WBC) and clinical disease progression.

Using flow cytometry, high numbers of circulating lymphocytes in S phase had a shorter therapy-free and total survival compared with those with fewer S-phase cells (65). Also, Orfao et al. (66) showed that a high absolute count of circulating S-phase leukocytes was associated with a higher incidence of hepatosplenomegaly, anemia, and thrombocytopenia, a higher number of lymphocytes in blood and bone marrow, advanced clinical stages, lower serum IgG and IgM, and poorer survival. Moreover, the fraction of circulating Ki-67-labeled cells in CLL correlated with the proportion of prolymphocytes and was higher in resistant CLL than in indolent cases (67). Even high proliferative in vitro responses to B-cell mitogens were significantly associated with poor survival, whereas unstimulated thymidine uptake did not predict outcome (62). Studies of lymphocyte doubling time (LDT) confirmed the above data. LDT is defined as the period needed for the peripheral lymphocyte count to double the original count and has been associated with prognosis. A low LDT (< 12 mo) is associated with a poor survival and predicts rapid disease progression (68).

Other proliferative markers such as PCNA concentration were significantly lower in earlier stage CLL, and the level of this marker, which correlates with proliferative phase and LDT, also correlated with known prognostic factors such as disease stage in CLL patients (69). Similarly, both the percentage and absolute number of Ki-67-expressing cells were found to increase with disease stage (70).

High expression of the cyclin-dependent kinase inhibitor p27, which contributes to cell cycle arrest, may also be a valuable kinetic marker in B-CLL, since stable CLL patients usually express low levels of p27, whereas progressive CLL patients show a significant overexpression of this cyclin-dependent kinase inhibitor (71).